Macrophages are integral components of the immune system that play a critical role in the identification, engulfment, and clearance of dead or dying cells in the body. Defects in this process can contribute to a wide range of diseases, including autoimmune disorders, neurodegenerative diseases, and cancer.

Thus, Creative Biolabs helps researchers to understand the mechanisms and factors that regulate the clearance of dead cells by macrophages and may provide therapeutic opportunities for modulating the immune response and the onset or progression of a variety of diseases.

Release of Find-me Signaling by Apoptotic/Dead Cells

There are two direct pathways, including the embedding of macrophages in tissues undergoing sustained apoptosis or the direct delivery of dead cells to macrophages through the circulation. However, in some cases, tissue-resident and circulating macrophages need to be recruited and migrate to the apoptotic cell region. Find-me signaling plays a key role in this process.

Fig. 1 Find-me signaling released by apoptotic cells recruits macrophages.¹

Injured and dead cells release signals that are recognized by macrophages that migrate to the area where the cells are located. The most studied signals found are

• Lysophosphatidylcholine (LPC)
• Sphingosine-1-phosphate (S1P)
• ATP, AMP, and UTP

Macrophages use a range of receptors to detect and respond to find-me signaling.

• G protein-coupled receptor (GPCR) G2A recognizes LPC.
• There are five S1P receptors in mice.
• Tissue macrophage populations express several different P2Y purinoceptors (P2Y2, P2Y6, P2Y12, P2Y13, and P2Y14) that bind ATP, ADP, UTP, and UDP.

Identification of Cell Surface Markers

Macrophage exposure to apoptotic/dead cells requires specific macrophage surface markers to be recognized, and the most prevalent, effective, potent and important is undoubtedly phosphatidylserine (PtdSer). The proteins that directly or indirectly bind to PtdSer on the surface of macrophages include T cell immunoglobulin and mucin domain-containing molecule (TIM), TAM (TYRO3, AXL, MER) family, and others.

Fig. 2 Macrophages recognize apoptotic cells through ligand‒receptor interactions.¹

• TIM4: TIM4 is a highly glycosylated single transmembrane protein with a short (42 residues) cytoplasmic tail. It binds tightly to PtdSer in Ca²⁺-dependent reactions (low nanomolar affinity), but not to phosphatidylcholine, phosphatidylinositol, or phosphatidylethanolamine.
• TAM receptors and their ligands: They are the most widely expressed PtdSer recognition systems in macrophages. The TAM proteins TYRO3, AXL and MER are cell surface receptor tyrosine kinases (RTK). These receptors do not bind directly to PtdSer, but rely on their activating ligands GAS6 and PROS1 for this activity.
• Other eat-me signaling and receptors: There are other receptors that play a role in PtdSer binding and recognition, including
  • CD300b, a protein that binds directly to PtdSer, uses the junction protein DAP12 for intracellular signaling, and mobilizes F-actin for phagocytosis.
  • BAI1 is an adhesion-associated GPCR that binds to PtdSer, cardiolipin, phosphatidic acid, sulfate, and LPS to promote phagocytosis of apoptotic cells.
  • Another direct binding PtdSer, specifically targeted for erythrocyte clearance, is stabilin 2. It is highly expressed in erythromedullary macrophages and is present in Kupffer cells, but it is not normally expressed in tissue macrophages.
• Don't eat-me signaling: Counteracting, phagocytosis-inhibiting activity, or so-called don't eat-me signaling as a negative regulatory mechanism for phagocytosis. The classic is CD47-SIRPα.

Macrophage Endocytosis of Apoptotic Cells

The key molecule in macrophage endocytosis of apoptotic cells is Rac1, whose activation leads to actin polymerization and induces cytoskeletal rearrangements to phagocytose the target. After phagocytosis of apoptotic cells, macrophages degrade them. The phagosome becomes increasingly acidic by sequentially recruiting Rab5 and Rab7 and eventually fuses with lysosomes containing digestive enzymes required for apoptotic cell degradation.

Phagocytosis of apoptotic cells brings multiple levels of complexity: in ingesting multiple apoptotic cells, the phagocyte needs to manage its volume and surface area, and the ingested cargo is a metabolic burden carrying membranes, cholesterol, proteins, nucleic acids, etc., which need to be metabolized and either reentered into the metabolic flux cycle of the phagocyte or excreted.

Creative Biolabs provides our clients with phagocytic capacity analyses of macrophages. By leveraging our wealth of information on macrophage characterization, we are pleased to offer customized services to help our clients with meaningful projects. For more information, please feel free to contact us and discuss further with our scientists.

Reference

1. Moon, Byeongjin, et al. "After cell death: the molecular machinery of efferocytosis." Experimental & Molecular Medicine 55.8 (2023): 1644-1651.